Process for extraction of antibiotic material



' Patented Apr. 24, 1951 PROCESS FOR EXTRACTION F ANTIBIOTIC MATERIAL Lloyd B. Jensen, Chicago, 111., assignor to Swift 8; Company, Chicago, 111., a corporation of Illinois No Drawing. Application April 5, 1946, Serial No. 659,881

The present invention relates to the preparation of a potent antibiotic substance, and it has to do more particularly with the concentration of an antimicrobial substance of plant origin.

For some time it has been known that some pathogenic bacteria are destroyed when soil is inoculated therewith. This destruction of bacteria is due to certain bacteria or antibacteria substances contained in the soil. It is also well known that the putrefying bac teria responsible for food spoilage do not ordinarily develop in living tissues of plants and animals. The presence of antibodies in live animal tissues and of. antibiotic substances in plants are thought to play an important part in establishing immunity in living organisms against microbes harmful to the organism. From time immemorial it was thought that certain medicines I of herbal origin were of some therapeutic value.

Pursuantto this knowledge, microbiologists have been seeking to isolate useful microorganisms and prepare antibacterial substances from natural sources which antibacterial materials, although disassociated from their environment, might be capable of combatting microorganisms harmful to man. Although living matter is known to contain substance of an antibacterial nature, it is impossible to predict whether or not portions of a living organism, for example a plant, will yield an effective antibacterial substance when subjected to known methods of separation, concentration, extraction, or purification; or if once isolated whether or not the antibacterial substance will be beneficial. As a result of this search for antibacterial agents, naturally occurring antibiotic substances have been prepared from plants, animal tissues, molds, actinomyces, yeast and bacteria, for example, penicillin from the, mold Penicillium notatum, and gramicidin from certain soil bacteria. It is known that some tain types of bacteria, they are, on the other hand, ineffective against other types or strains of bacteria.

An object of the present invention is to provide a potent antibiotic substance of plant origin. A further object of the invention is to provide an antibiotic substance which is not toxic.

5 Claims. (cum-s5) A still further object of the invention is to provide an antibiotic substance which'is heat stable.

Yet another object of the invention is to provide an antibiotic substance which may be employed as an effective preservative for food products.

Still another object of, the invention is to pro vide an antibiotic material which is capable of inhibiting mold growth.

Other objects and advantages ofthe invention will be apparent from a description of the invention given below. V

The present invention contemplates broadly the preparation of an antibiotic substance found to be naturally associated with tissues of 'the' tree Persea gratz'ssima, or Persea americana, commonly known as the avocado tree, including the fruit thereof. I have discovered that especial ly the pit or pith of the avocado fruit or pear yields an antibiotic substance of great potency, to be hereinafter more fully described. I have also found that the antibiotic substance produced from the av'ocadopit is lethal to those microorganisms largely responsible for clostridial and bacillary food spoilage, and staphylococcus food poisoning, and therefore may be used advane tageously in the preservation of food products. My antibiotic substance has been found to destroy gasforming bacilli of the genus Bacillus, such as Bacillus subtilis' (var. nicer) molds of the genera Aspergillus and Penicillium, such as Aspergillus glaucus, and Penicillz'um notatum; the bacilli of the genus Clostridiunigsuch as C'Zostridium sporogenes, the staphylococci, such as Staphylococcus aureus. I have also found my antibiotic substance to be lethal against the bacterial species Achromobacter perolens, the organism responsible for the production of a musty odor in eggs known as hay eggs. .Whilethe antibacterial substance produced from the avocado pit is extremely e'fg. fective against the aforesaid. organisms, I have found that other organisms, for example, 'E sg and varieties of microorganisms to the herein decherichz'a coli, Penicillium cmnem berti, the

Pseudomonas fluorescens and Pseudomonas syncyomea are more resistant thereto. an extremely arduous, if not an impossibletask to ascertain the reponse of all the existing species scribed antibiotic material but the effects of this material" against the microorganismshereinabove' It would be mentioned illustrate the commercial and medical values thereof.

The invention contemplates treating a mix ture of ground avocado piths or pits with a suitable solvent to recover the antibiotic substance from the avocado material. A colored powder remains after evaporating off the solvent and may advantageously be refined by treating again with a suitable solvent, such as ethanol.

The following examples illustrate preferred embodiments of the invention, and the tests falling thereunder illustratethe potency of the material obtained': in accordance with the processes of the examples.

Example 1.-250 g. of avocado piths or pita (brand Ca1avo) were peeled and ground finely in a small hand meat grinder. The pits were cream colored with a slight yellowish tinge when whole, but upon grinding the: piths. became redorange in color and emitted a faint pleasantodor somewhat like a mixture of cinnamon and pine needles. The ground piths were covered with 500 cc. of acetone and held in a flash for 24 hours, the flash being shaken at intervals. The acetone solution was removed from the pith by filtration and the. acetone was distilled off under a.29inch vacuum. at.4'5 C- The. yellowish brown. residue was. dissolved in.50 cc. of 95. per cent. ethanol, so as to. remove the. possibility of solvent toxicity and. to prevent contamination. The alcohol also provides means for accurately measuring the strength of. the antibiotic substance; thus. the useof 50 cc. of alcohol makes. possible the establishment of an arbitrary standard. unit of antibiotic substance, that is, L ml. of alcoholic extract to represent 5 g. of original. plant. material.

Two tests' were then performed to evaluate the effectiveness of. the product obtained in. accordance with the process described. in Example I.

Test l.--One hundred milliliters of nutrient agar (obtained from Difco Laboratories, Detroit, Michigan) were inoculated with 0.2 ml. of a 20-hour broth culture of a. food poisoning strain of Staphylococcus aareas, which had been tested. on Macacus rhesus monkeys and found to produce enterotoxin. Plates were poured with this agar. A hole was boredin the center of the hardened agar so as to form a cup in the agar plates. The cups were then filled with aqueous dilutions of the antibiotic product (1 ml. of the product representing 5. g. of pith) and were incubated at 37 C. for 4 days. Although theinoculum was rather heavy so as to prevent good diffusion thereof through the agar, the areas of. inhibition indicated in the. following table were obtained.

The control plate was prepared by pouring. agar therein,v and after. the agar had. hardened, a. cup was formed. by boring. the. agar. The cup. was then filled with a 12 100 aqueous dilution. of. 95%. ethanol In the control plate, coloniesof the Staphylococci were. observed throughout the agar, while in the other plates containing the antibiotic material growth of bacteria began to be manifested at varying distances away from the edge of the cup. Thus in a 1:10 dilution, growth of the bacteria was first noticed 9 mm. from the edge of the cup; at 1:50 dilution, 5 mm. away; 1:100 dilution, 4 mm. away, etc. Presumably, the clear zone of inhibition characterized by the absence of bacterial growth represented the extent to which the antibiotic material had diffused through the agar.

The agar cup plate method. is open to objections in evaluating the; potency of an: antibiotic or other antibacterial substance because the method depends in part upon rate of diifusion of' the substance through the agar. Serial dilution. methods in nutrient broth tubes, as described below, were then employed to demonstrate antibiotic action on bacteria.

Test 2I..In. this test the potency of the antibiotic substance obtained from Example I was determined by serial dilutions of the extract in 10 cc. broth tubes and then inoculated with bacterial. Dilutions of the extract were made in broth tubes from 1: 10 to"l:80,000 and inoculated with large loops of 24-hour broth cultures of a food. poisoning strain of Staphylococcus aureas (50,000 cells). The test organism was inhibited at a dilution of 125000 after 7 days incubation at 37 C. After 7 days. all of the tubes showing no growth were used as inocula in nutrient agar plates, 0.1 cc. of the material in the broth tubes being transferred to each agar plate. No growth resulted in'3 days from these transfers, which. shows that the antibiotic substance had killed the test" bacteria in a 1:5000 dilution.

Example II.'-340 g; of avocado piths (Rancho-La Habra brand) were groundunder acetone and covered with acetone for 24 hours. The solvent was evaporated off under 27 inches of vacuum (water pump) at 40 C. and the residue taken up in 68 ml. of pure ethyl alcohol, so that each ml. of alcoholic solution represented 5 g. of piths.

The effectiveness of the bactericidal properties of the product obtained by means of the process described in Example II, was then tested according to the method of Test 2 of Example I; that is, serial dilutions of 10cc. broth tubes were made, each tube inoculated with a large loopful of a broth culture of Staphylococcus aureus incubated for 24' hours at 37 C. The inoculated tubes were then allowed to incubate at 37 C. and were observed for growth at the end of 24, 48, and hours. The following table gives the dilutions and results as to bacterial growth corresponding therewith after 24, 48, and 120 hours, the'minus sign indicating no growth, the positive sign the presence of growing staphylococci.

The tubes of incubate at 37 the above test were allowed to C- for 7 days. At the end of 7 days, the tubes up to and. including 125000 dilutions showedv no growth, and were used as inocula on nutrientagar plates by transferring into the agar plates 0.1 cc. of thematerial contained in the dilution tubes. After incubation for 3 days, no'growth resulted in the agar plates, thus indicating that the antibiotic substance was an effective bactericide in 1:5000 dilutions. In dilutions higher than 125000, the antibiotic substance shows antiseptic or bacteriostatic propciation strain"#3679-) was killed in a l:10,000'

dilution in Difcopeptone colloid tubes; species of genus Bacillus in 1:500 dilution in Difco nutrient broth; Achromcbacter perolens, Aspergillus' gl ucus and Penicillium notatum also in 1:500 dilution, and another strain of Staphylococcus aureus in 1: 10,000 dilution.

-Although I have found that the pits of the fruit of the avocado tree are especially rich in a powerful antibiotic substance, other parts of the tree, such as the leaves, stalk, roots and meat of the fruit contain the antibacterial substance to a lesser extent, .but sufiiciently to show bacteriostatic, or antiseptic, properties.

- I The antibiotic substance contained in Pe'rsea gratisstma is soluble in a number of solvents. Suitablesolvent media are the polar solvents, such as acetone, methyl ethylketone, ethyl, propyl and butyl alcohols, dioxane, ethyl acetate; chlorinated solvents, such as carbon tetrachloride, trichloroethylene; hydrocarbons, such as pentane, hex-' ane, etc." Sincethe antibiotic material isolated from the avocado plant isheat stable, solvent treating temperaturesmay range from room temperates to boiling temperature of the solvent. The type of solvent chosen affects the color of the dried product. For example, acetone gives a brownish red product; hexane and trichloroethylene, a yellow material; ethyl alcohol, methyl ethyl ketone, ethyl acetate, a red substance, etc. It is obvious that many variations in the solvent treatingprocess may be advantageously em- PIOYGCL' To illustrate, the treatment may be carried out in one step, for example, with ethanol;

mixtures of solvents and different temperatures may also be advantageously employed to increase elficiency. I have found it preferable to process the antibiotic substance contained in the avocado pit by a two-stage solvent process, involving, first, treating the substance by means of a primary solvent which is subsequently removed, and then,

second, taking up the resulting residue with another solvent, so as to form a solution, which is a convenient form of using the product. I have found that acetone isa very effective primary solvent in producing the antibiotic substance" immediately. If the alcoholic solution in broth or water is centrifugalized and dilutions made in broth tubes of both supernatant liquid and sediment which are then inoculated with bacteria, both supernantant liquid and sediment inhibit bacterial growth. For example, a 1:10 dilution.

of the alcoholic solution in broth was centrifu- By using alcohol I may utilize itswell known properties, such as nontoxicity and ability g a1ized'30 minutes at'high, speed and dilutions made in broth tubes of both supernatant liquid and sediment. The tubes (10 ml.) were each inoculated with a loopful of a 20-hour broth 5: culture of food poisoning Staphylococcus aureus and incubated at 37 C. Bacterial growth at 1:100, 1:500, 1:1000, 1:2000, 125000 and 1:7500 dilutions of the extract were observed after 24 and 48 hours. The following table lists the re- 10: sults, the minus sign indicating no growth, the

positive sign indicating bacterial growth.

Table III s t 1; P 't t 15 a star star Dilution 24m. 48l1rs. 24m. 48 hrs.

I I I I The data tabulated above show that the precipitate is slowly soluble in water and is a powerful antibiotic substance.

In the processing of food products, a serious problem is that of food preservation, and although great strides have been made in improving the quality and keeping time of various food prod ucts by refrigeration, canning, curing, plant sanitation, etc., the problem of the preservation of food requires the constant attention of the food processor to improve long-established methods of treating food and to devisenew means of food 1 control.

Food spoilage is due principally to microbial action, and the microorganisms responsible for food deterioration are numerous. The sporing genera, Clostridium and Bacillus, for example, when contaminating food, cause food spoilage. Molds of the genera Penicillium and Aspergillus are also responsible for much food spoilage. In addition to food spoilage, a serious concern of the food handler is food poisoning, which is often of an insidious nature because food products contaminated with harmful bacteria often appear organoleptically sound and Wholesome. Among S- the food poisoning bacteria, the most common food poisoner is the staphylococcus. This organism is Widespread in nature, and is believed to be responsible for over 90 per cent of food poisoning cases in the United States, in addition to its l i being responsible for many infections in man and animals.

The antibacterial substance of the present invention may be used to retard or prevent micro- 60 bial growth in various food products. This material may advantageously be employed in treating meat products, such as hams, sausages and canned meats; filled pastries, such as cream puffs, chocolate eclairs, custards, etc.; canned vegetables to prevent swellings due, for example, to

K1 organisms of the genus Bacillus.

The invention will be more fully understood from the following description of a method of using my antibiotic substance, in connection with the curing of hams.

1 Within recent years there has been developed acuring process for meats and particularly hams known as a quick-cure which requires only a fraction of the time which was normally required by the old processes. Essentially the quick-cure processconsists in pumping the pickling soluthemeat; By: this means: a. ham.can;be;;c.uredu inaperiod of time rangingifrom fifteenito eight..- een days, rather than: the sixty to. ninety days, required-2 by the old .processes::in: which. the .meats were :usually. immersed in a bath of 1 the pickling;

There-haszrisen, however; as the result of (ex-- tensive commercial useof the quick-curing method an. attendant'serious.health problem.: The hams and-other. meats curedby the older immersion processes wereextremely resistant to the development of pathogenic bacteria, of which various representatives of the Staphylococci may be mentioned as typical, and the general public has come to rely onthis property in its eatin and storage habits. Hams processed by quickcuring; on the other hand, have resulted in a. meat product which, though highly satisfactory. when fresh, is not nearly so resistant to these organisms, and numerous cases of food poisoning have resulted from the general publicsunfamiliarity with thisfact.

As hereinbefore mentioned, we have found that the antibiotic reagent herein described. islethal to a number of microbes in dilution of 1:5000, based on the arbitrary unitthatl ml. of solution is equivalent to 5 g. of the source material, or original pith. Therefore, the addition of one part of the solution to 5000parts of the conventional pickling solution will result in a meat prodnot cured therewith which has a very marked increase in resistance to. the development of pathogenicorganisms.

The followingis an example of a curing pickle stock, which may be usedin a process for quick curing ham when'provided with antibioticsubstance in the proportions outlined above; that is,

the antibiotic substance is added in an amount.

such as to effect a.1.:5000 dilutionof the-antibiotic material, based on the arbitrary standard herein described (1 ml.=5 g. of pith) To 100 gallons of-a-90f Salometer solution there are added:

'7 lbs; sodium'nitrate oz. sodium nitrite lbs. sugar '75.? cc. antibioticsolution The following. experiments will. further illustrate the practical application of my invention; An amount of pickle was inoculatedwith alarge number of a foodpoisoning type of Staphylococcus amreas organisms which. wouldrepresent the most unfavorable conditions which could possibly be encountered. The pickle was then separated: into two equal portions, and to one portion sufiicient amountof avocado product was: added toform a 1:500!) dilution of. the product; Togthe other portion of pickle no antibiotic-substance was added. Twelve hams were then: cured: by' the quick-curing. method-six of them. withpickle containing; the antibiotic substance. and" six with pickle without the substance. The

hams were then smoked accordingto ordinary" processes. The hams were incubated at 99? F. and at theend of; two weeks the six hams containing the antibiotic treated'hams did notharbor viable staphylococci, while. the other hams showed thepresence of'viable staphylococci.

Luncheon meat was cured according. to conventional. methods in which the antibioticsub. stance was. present in a 1:5000 dilution. The meat product wasplaced in 6 six-pound cans and retorted .to; an inside. temperature 1of;l559 and also incubated: atQ9i-F. A"fter: days of incubation, no swelling of the;cansr could L be observed;

The .controlcans swelled in one week.

A dry acetonesolutionof product from 60 g. 5:. of"avocado:pith was1fed to four white rats for six: days. The animals; consumed 600 g. of ground meat to" Whichwas added 1 per cent of the-product. The animals-showed no'symptoms,

of toxicity and-there-Were-no' losses in, weight;

Obviously, many modifications and variations of the invention,- as hereinbefore-set forth, may

be made. without departing from: the spirit and lot scopethereofland therefore only such limitations.

shouldbe imposed aszare indicated. in: the am;- 15.; pendedclaims.

1. A process of preparing; a powerful anti-' biotic. substance: which comprises comminuting thefruit pit of thetreePersea gratissima, treat- 20: ing the said fruit pit'with acetone:ata temperature not substantially, in excess of roomtemperature. to form.-a;non-.a queous solution of theantibiotic material, separating. the solution from the insoluble residue, andpreducing the solution to 25:- dryness by subjecting the saidsol'utionto distillation at a reduced-pressure to obtain. aipowerful antibiotic substance;

2. A' process of. preparing a powerful antibiotic substance which comprises comminuting the fruit pit of the tree'Perse a gratissima, extracting the said fruit'pitwith acetone at about room temperature to form a non-aqueous solution of theantibiotic substance, separating the said solution from theacetone; insolubleresidue, and reducing theso-lutionto dryness by distilling at a.reduced.-pressure to obtainia-powerful anti biotic material;

3. A, process of preparing as. powerful antibiotic substance which comprises. comminuting the fruit pitof the tree- Persea gratissima, extracting the said" fruit pit-.at about. room temperature with at least approximately twice its weight of acetone-to remove substantially all the acetone. soluble materialfrom thesaid fruit pit and form a substantially non-aqueous. solution of the antibiotic; substance,. separating the said solution from the acetone insolublematerial, and reducing the saidsolutionto dryness by subjecting the solution to distillation at a reduced pressure substantially, below atmospheric pressure and at a temperature substantially below the boiling point of acetone at atmospheric pressure to forma dry powerful antibiotic substance.

4. The process of preparing a powerful antibiotic:v substance which comprises comminuting the fruit pit of thetree Persea gratz'ssima, ime mersing the saidfruit pit inabout twice its weight of acetone at about room temperature and allowing the said fruit pit to stand in said acetone for approximately 24 hours with intermittent agitation to remove substantially all the acetone soluble material of the said fruit pit and form a substantially non-aqueous solution of the antibiotic substance, separating. the said solution from the acetone insoluble material, and reducing the said solution to .dryness by subjecting the.

said solution to distillation under a high vacuum and at a temperature substantially below theboiling point of acetone at atmospheric pressure to 1 form a dry powerful antibiotic material.

5. A process substantially as in claim-.4 wherein the Vacuum is about 29 inches of mercury and the temperature is about 45C.

LLOYD B. JENSEN.

(References on following page) 9 10 REFERENCES CITED Number Name Date 4 2,241,030 Hess May 6, 1941 3 ififjggfii i record m the 2,399,340 Wachtel May 7, 1946 UNITED STATES PATENTS 5 OTHER REFERENCES Number m Date Bilger et 21.: Annual Report Hawaii Agr. Exp.

1,417,771 Schamberg et a1. May 30, 1922 ,91 4-17 2). 1,635,302 Alsberg July 12, 1927 E 3 Bra$- Farm, 2,084,864 P d k J 2 19 7 ad 00 um 3 Khouvme et 21.: Compt. rencL, vol. 206, pp.

1 2,131,394 Test Sept. 27, 1938 10 2,180,750 Urbain Nov. 21, 1939 (1933)- 

1. A PROCESS OF PREPARING A POWERFUL ANTIBIOTIC SUBSTANCE WHICH COMPRISES COMMINUTING THE FRUIT PIT OF THE TREE PERSEA GRATISSIMA, TREATING THE SAID FRUIT PIT WITH ACETONE AT A TEMPERATURE NOT SUBSTANTIALLY IN EXCESS OF ROOM TEMPERATURE TO FORM A NON-AQUEOUS SOLUTION OF THE ANTIBIOTIC MATERIAL, SEPARATING THE SOLUTION FROM THE INSOLUBLE RESIDUE, AND REDUCING THE SOLUTION TO DRYNESS BY SUBJECTING THE SAID SOLUTION TO DISTILLATION AT A REDUCED PRESSURE TO OBTAIN A POWERFUL ANTIBIOTIC SUSBTANCE. 